Hydrogels loaded with chemotherapeutics are promising tools for local tumor treatment. In this work, redoxresponsive implantable hydrogels based on gellan gum were prepared as paclitaxel carriers for HER2-positive breast cancer therapy. To achieve different degrees of chemical crosslinking, hydrogels were synthesized in both acetate buffer and phosphate buffer and crosslinked with different concentrations of L-cysteine.
Trang 1Available online 15 June 2022
0144-8617/© 2022 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/)
Biodegradable gellan gum hydrogels loaded with paclitaxel for HER2+
breast cancer local therapy
Celia Nietoa, Milena A Vegaa, Víctor Rodrígueza, Patricia P´erez-Estebanb, Eva M Martín del
Vallea,*
aChemical Engineering Department, Faculty of Chemical Sciences, University of Salamanca, Salamanca 37008, Spain
bCollege of Health and Life Sciences, School of Biosciences, Aston University, Birmingham B4 7ET, UK
A R T I C L E I N F O
Keywords:
Gellan gum
Hydrogel
Local chemotherapy
HER2-positive breast cancer
Paclitaxel
β-Cyclodextrin
Glutathione
A B S T R A C T Hydrogels loaded with chemotherapeutics are promising tools for local tumor treatment In this work, redox- responsive implantable hydrogels based on gellan gum were prepared as paclitaxel carriers for HER2-positive breast cancer therapy To achieve different degrees of chemical crosslinking, hydrogels were synthesized in both acetate buffer and phosphate buffer and crosslinked with different concentrations of L-cysteine It was shown that both, the type of buffer and the L-cysteine concentration used, conditioned the dynamic modulus, equilibrium swelling rate, porosity, and thermal stability of the hydrogels Then, the biocompatibility of the hydrogels with the most suitable porosity for drug delivery applications was assessed Once confirmed, these hydrogels were loaded with paclitaxel:β-cyclodextrin inclusion complexes, and they showed a glutathione-
responsive controlled release of the taxane Moreover, when tested in vitro, paclitaxel-loaded hydrogels
exhibi-ted great antitumor activity Thus, they could act as excellent local tailored carriers of paclitaxel for future, post- surgical treatment of HER2-overexpressing breast tumors
1 Introduction
Breast cancer is currently considered as one of the diseases with the
highest mortality rate in woman worldwide (Tang et al., 2021), with
685,000 deaths associated with female breast cancer being reported last
year alone (Sung et al., 2021) Among the different alternatives that
exist for its treatment, surgical resection is the gold standard clinical
strategy (Bu et al., 2019; Tang et al., 2021; Zhuang et al., 2020)
Nevertheless, despite much improvement in surgical techniques,
effi-cient inhibition of breast cancer recurrence still presents a challenge
The main reason for this is that residual tumor cells can remain in
sur-gical margins (Askari et al., 2020; Bastiancich et al., 2017), particularly
in patients who have undergone breast-conserving therapy (Qu et al.,
2015)
To reduce the incidence of relapse, radiotherapy and chemotherapy
are routinely administered in the clinical setting after tumor resection
However, both treatments are associated with high toxicity and severe
systemic side effects (Bu et al., 2019; Tang et al., 2021) In addition,
since these forms of treatment must begin in the weeks following surgery
to allow the patient's health to recover, residual infiltrative cancer cells
can keep proliferating in the meantime (Bastiancich et al., 2017; Bu
et al., 2019; Zhuang et al., 2020) Moreover, resistance to chemotherapy may be promoted, in addition to other factors such as hypoxia or al-terations in the signaling pathways of cancer cells, by the limited tar-getability of the anticancer drugs (Askari et al., 2020; Kibria & Hatakeyama, 2014) For these reasons, local delivery of chemothera-peutics in the tumor resection cavity is becoming increasingly desirable for breast cancer treatment (Tang et al., 2021) Compared to systemic therapies, local chemotherapy can prevent drugs from being non- specifically distributed and can avoid off-target toxicities Moreover, local chemotherapy may eliminate the latency time of post-surgical systemic chemotherapy (Askari et al., 2020; Tang et al., 2021; Zhuang
et al., 2020)
Among the different types of drug delivery systems (DDS) designed for antitumor local therapies, hydrogels are, in particular, generating greater interest, as their mechanical properties can be tailored to mimic those of the extracellular matrix (ECM) of living tissues (Askari et al.,
2020) Furthermore, most of these three-dimensional hydrophilic net-works are made from natural polymers; thus, they are biocompatible, biodegradable and easily modifiable, in addition to having high drug-
* Corresponding author
E-mail address: emvalle@usal.es (E.M Martín del Valle)
Contents lists available at ScienceDirect Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
https://doi.org/10.1016/j.carbpol.2022.119732
Received 3 February 2022; Received in revised form 30 May 2022; Accepted 9 June 2022
Trang 2loading capacities (Abasalizadeh et al., 2020; Darge et al., 2019; Misra &
Acharya, 2021; Sharma & Tiwari, 2020) Among the most common
natural polymers, gellan gum (GG) is gaining attractiveness for
biomedical purposes, as it is stable and has appropriate mechanical
properties, acid and heat resistance and inotropic sensitivity GG is an
extracellular polysaccharide, which contains repeating units of β-D-
glucose, L-rhamnose, and D-glucuronic acid in a 2:1:1 M ratio (Das &
Giri, 2020; Palumbo et al., 2020), that can undergo thermally reversible
gelation after a coil-helix transition in the presence of mono- (K+, Na+)
or divalent (Ca2+) cations (Bacelar et al., 2016; Prajapati et al., 2013;
Soleimani et al., 2021) Similarly, GG can be chemically crosslinked to
maintain stable biomaterial structures for longer periods Previous
works involving this polysaccharide have been reported regarding its
use for the delivery of several anticancer drugs (paclitaxel, doxorubicin,
erlotinib and clioquinol, among others) to improve their solubility,
intra-tumoral specificity, and drug release profile via hydrogels, patches
and nanoconfigurations (Villareal-Otalvaro & Coburn, 2021) In the
specific case of paclitaxel (PTX), GG has been employed to develop in
situ-gelling liposome-in-gel composites containing this drug for local
bladder cancer treatment, and nanohydrogels delivering the taxane
along with prednisolone for prostate cancer and inflammatory
carci-noma applications (D'Arrigo et al., 2014; GuhaSarkar et al., 2017)
However, GG has not yet been used to fabricate PTX-loaded implantable
hydrogel patches for local, stimuli-responsive treatment of HER2-
positive (HER2+) breast tumors Therefore, the main aim pursued in
this work was to develop, characterize and validate in vitro PTX-
releasing GG hydrogel patches that would be suitable for this novel
application: local and redox-responsive antitumor therapy of HER2+
breast tumors
Consequently, GG hydrogels (HGGs) were prepared in two solutions
with different pH and ionic compositions (acetate buffer [AB] vs
phosphate buffered saline [PBS]) and were disulfide-crosslinked with
different L-cysteine (L-Cys) concentrations utilizing the carbodiimide chemistry to improve their stability while achieving responsiveness to external reducing stimuli, such as the high glutathione (GSH) concen-trations existing in malignant breast cells (Li et al., 2020; P´erez et al.,
2014) The main aim of synthesizing HGGs in different buffers and with different L-Cys concentrations was examining how these parameters conditioned their crosslinking degree and, therefore, their dynamic modulus, equilibrium swelling rate, porosity, and thermal stability Then, all these hydrogel properties were analyzed and, based on the results obtained, those HGGs with the most appropriate characteristics for drug delivery applications were selected to be loaded with PTX This taxane was previously included in β-cyclodextrin (βCD) molecules to improve its limited aqueous solubility (Nieto et al., 2019; Tian et al.,
2020), and the resulting complexes (PTX:βCDs) were included in the GG patches to enhance the redox-controlled release of PTX while trying to improve its bioavailability and off-target toxicity through a potential local application (Scheme 1) Antitumor activity of the HGGs loaded
with the PTX:βCD complexes was evaluated in vitro after analyzing their
biocompatibility, and the results obtained showed that they may be a promising strategy for post-surgical chemotherapy of HER2- overexpressing breast tumors with elevated GSH intracellular concentrations
2 Materials and methods
2.1 Materials
Gelzan™ CM (G1910, average molecular weight: 1000 kg/mol; low- acyl [0.2 %]; monosaccharide composition: β-D-glucose:L-rhamnose:D- glucuronic acid [2:1:1]), β-cyclodextrin (βCD, minimum 98 %),
pacli-taxel (PTX, from semisynthetic, >97 %), L-cysteine (L-Cys, 97 %), lyso-zyme human, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC),
Scheme 1 Schematic representation of the preparation of the HGG patches, chemically crosslinked with different concentrations of L-Cys and loaded with PTX:β
CD complexes
Trang 3N-hydroxy succinimide (NHS), thiazolyl blue tetrazolium bromide
(MTT), phosphate buffered saline (PBS, powder [NaCl [137 mM], KCl
[2.7 mM], Na2HPO4 [10 mM], KH2PO4 [1.8 mM], pH 7.4) and L-
Glutathione reduced (>98 %) were all obtained from Sigma Aldrich (St
Louis, MO, USA) Dimethyl sulfoxide (DMSO, >99 %) and Corning™
penicillin/streptomycin solution (100×: penicillin [100 UI/ml] and
streptomycin [10,000 μg/ml]) were purchased from Thermo Fisher
Scientific (Waltham, MA, USA) Calcein AM and propidium iodide (PI,
Ready Probes™) were obtained from Invitrogen (Carlsbad, CA, USA)
Potassium bromide (for IR), acetic acid glacial, citric acid anhydrous,
sodium acetate anhydrous, sodium citrate, sodium chloride, tris
hy-drochloride and absolute pure ethanol (EtOH) were all obtained from
Panreac AppliChem (Castellar del Vall`es, Barcelona, Spain) Dubelcco's
Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS,
quali-fied, HI) were purchased from Gibco (Gaithersburg, MD, USA) Finally,
lactate dehydrogenase activity colorimetric assay kit (product code:
ab102526) was obtained from Abcam (Cambridge, UK)
2.2 Synthesis of HGG patches
To prepare the HGG patches, Gelrite® (Gelzan™) was chosen among
the main different commercial forms of GG because it disperses and
hydrates well in deionized water (H2O[d]) and is inert to most biological
growth media additives (Prajapati et al., 2013) In this way, Gelzan™
was dissolved (1.5 % [w/v]) both in 80 ◦C AB (0.05 M, pH 4.0) and in
80 ◦C PBS (pH 7.4) (Matricardi et al., 2009; Oliveira et al., 2016) Once
homogeneous solutions were obtained, the temperature was lowered to
50 ◦C Solutions of EDC (2.9 mg/ml) and NHS (4.8 mg/ml) were later
incorporated consecutively (1:50 [v/v]) After stirring briefly, L-Cys
solutions of different concentrations (1.5, 3, and 4.5 mg/ml) were added
(1:50 [v/v]) to achieve different degrees of GG chemical crosslinking
(Wu et al., 2018; Yu et al., 2020) Final solutions were poured into dishes
and left for gelation at room temperature overnight
2.3 Rheology
Rheological measurements of 2-mm-thick HGGs were performed
using an AR 1500 Ex rheometer (Waters Corporation, Milford, MA, USA)
equipped with an aluminum parallel plate geometry (plate diameter 40
mm, gap distance 1 mm) HGG samples were prepared using 33 mm-
diameter dishes as templates, carefully unmolded preventing breakage
and placed on the lower plate of the rheometer To evaluate their
stiff-ness, dynamic oscillation-frequency tests were carried out in duplicate
in the 0.01–10 Hz range at 25 ◦C and 37 ◦C by applying a γ = 0.01
constant deformation in the linear viscoelastic region This region was
preliminary assessed using stress sweep tests (Matricardi et al., 2009)
(data not shown)
2.4 Swelling test
The swelling ability of the different HGGs was assessed via a general
gravimetric method Variations in weight were recorded over time when
the HGGs were soaked in solutions of different pH and ionic strength:
H2O(d) (purified with the Economatic Wasserlab equipment [Barbat´ain,
Navarra, Spain]); commercial mineralized water (H2O[c]); NaCl
solu-tion (0.015 M); tris buffer (0.05 M); citrate buffer (0.1 M); AB (0.04 M);
PBS (1×); and DMEM supplemented with FBS and antibiotics Briefly,
after gelation, hydrogel disks (35 mm diameter, 8 mm height) were
frozen at − 80 ◦C, lyophilized overnight (LyoQuest lyophilizer, Telstar,
Lisbon, Portugal), and weighed Then, hydrogels were immersed in the
previously mentioned solutions (50 ml), removed after different time
points, wiped superficially with bibulous paper, weighed again, and
introduced in the same solutions (Coutinho et al., 2010; Li et al., 2021;
Morello et al., 2021) The swelling ratios at time t (Qt) and when HGGs
reached equilibrium (Q∞) were defined according to Eqs (1) and (2),
respectively, where m0 is the initial weight of the dried gels (g), mt is the
weight of the swelled gels after time t (g), and m∞ is the weight of the swelled gels at the equilibrium (g) (Schott, 1992)
Q t=m t− m0
Q∞=m∞− m0
To better describe the swelling behavior of the HGGs, a swelling kinetic study was performed at the initial stage of swelling, until hydrogels reached equilibrium For this purpose, Eqs (1) and (2) were adjusted to a pseudo-second-order kinetic model, as described by Schott
in 1992 (Supplementary Material) It was considered that homogeneous uptake of the solutions occurred throughout the hydrogel polymer networks
2.5 Evaluation of the crosslinking density
The effective crosslinking density (dx, mol/ml) of the six different prepared HGGs was determined according to Eq (3):
d x= 1
where ϑ is the specific volume of the polymer (ml/g) and Mc is the average molecular mass between crosslinkings (g/mol), which was determined by the Flory-Rehner equation (Eq (4)):
M c= ρ p V s V 1/3
r
[
ln(1 − V r) +V r+XV2
r
where V s is the molar volume of the solvent (ml/mol), ρ p is the density of the polymer (g/ml), X is the parameter of interaction between the sol-vent and the polymer (which has a value of 0.81 ± 0.05 for aqueous solutions of GG (Safronov et al., 2019) and Vr is the polymer volume fraction calculated from Eq (5)
V r=
[
1 +ρ p
ρ s
(
M a
M b
) +ρ p
ρ s
]
(5) where Ma is the swollen hydrogel weight (g), Mb is the weight of the dried hydrogel before the swelling experiment (g) and ρ is the density of the solvent (g/ml) (Afinjuomo et al., 2019; Sabadini et al., 2018)
2.6 Morphological analysis and porosity determination after freeze- drying
The porous structure of the different HGGs synthesized was analyzed
by scanning electron microscopy (SEM) (ESEM Quanta 200 FEG, FEI, Hillsboro, OR, USA) HGG samples were freeze-dried, coated with gold and cross-sectioned Then, samples were imaged at an accelerating voltage of 15 kV 8 to 10 images were acquired from different areas of each sample and the average diameter of the micro- and macropores
existing in the HGGs was determined via image analysis (ImageJ
soft-ware) (Hua et al., 2016; Lee et al., 2020)
In addition, HGG porosity was measured using Archimedes' princi-ple Once synthesized, all hydrogel samples were freeze-dried and completely immersed in tubes filled with absolute EtOH After 24 h, HGGs were removed from the tubes and their porosity was calculated according to Eq (6):
Porosity (%) = W2− W3−W s
W1− W3
where W1 is the weight of the tube filled with EtOH (g), W2 is the weight
of the tube filled with EtOH 24 h after immersion of the freeze-dried HGGs (g), W3 is the weight of the tube filled with EtOH after HGG removal (g) and WS is the weight of the freeze-dried HGGs (g) (Goodarzi
et al., 2019)
Trang 42.7 Fourier transform infrared (FTIR) characterization
The chemical structure of all HGG samples, as well as that of GG, was
analyzed by FTIR spectroscopy (Spectrum Two™ spectrometer, Perkin
Elmer, Waltham, MA, USA) at the wavelength range of 900–4000 cm− 1
and compared Freeze-dried samples were ground to powder, dried at
37 ◦C for 3 days to remove any possible residual water, prepared with
KBr pellets, and scanned
2.8 Thermogravimetric analysis (TGA)
HGG thermal stability was analyzed by TGA (DSC Q100 calorimeter,
Waters Corporation, Milford, MA, USA) and compared to that of GG
alone HGGs were freeze-dried, and all samples were later ground to
powder and heated at a rate of 10 ◦C/min from 50 ◦C to 600 ◦C under a
nitrogen atmosphere to obtain the thermogravimetric (TG) curves
2.9 Compression test
The compressive modulus of cylinder samples (35 mm diameter, 8
mm height) of the HGGs chosen to be later loaded with the PTX:βCD
complexes was determined by spherical indentation testing Thus, a
spherical indenter was employed as plunger (Fig S1), the force-
indentation curve for the samples was recorded, and the effective
stiff-ness of the hydrogels was extracted For this purpose, the indentation
curves obtained were fitted to Hertz's contact model (Eq (7)) (Srivastava
et al., 2017)
F = − 16E2
̅̅̅̅̅̅̅̅
Rd3
√
where F was the force applied by the indenting bead (N), E2 was the
Young's modulus of the different HGG samples (kN/m2), R was the
diameter of the bead (6 mm) and d was the indentation depth (mm)
HGG average Young's modulus was determined from the slope obtained
after plotting F vs d3/2 Three parallel samples were tested to obtain an
average
2.10 Hydrogel in vitro degradation
The degradation rate of the HGGs (35 mm diameter, 8 mm height)
later loaded with the PTX:βCD complexes was investigated in vitro
through weight loss under simulated tumor extracellular pH conditions
Once weighed (m0, g), HGGs were placed in duplicate in beakers
con-taining lysozyme solution (1 mg/ml in PBS (pH 6.8)) and incubated for
9 days at 37 ◦C under gentle shaking (50 rpm) HGGs were weighed daily
(mt, g) after wiping their surface with bibulous paper, and their weight
loss (mr) was determined according to Eq (8) (Huang et al., 2020; Lu
et al., 2022; Panczyszyn et al., 2021; Xu et al., 2018):
m r(%) =m0− m t
m0
2.11 Cell culture and hydrogel biocompatibility in vitro
Human HER2+ breast carcinoma BT474 cells and stromal HS5 cells
were grown in DMEM supplemented with 10 % (v/v) FBS and 1 % (v/v)
penicillin/streptomycin, and cultured in an atmosphere of 5 % CO2 at
37 ◦C
HGG biocompatibility was doubly assessed by MTT assays and live/
death staining BT474 and HS5 cells were seeded in 24-well plates
(12,000 cells/ml), grown for 24 h for attachment, and cultured with
HGG samples that were allowed to gel for 90 min (23.1 % [v/v],
pre-viously sterilized by UV radiation) Cells were incubated for 72 h and
their survival rate was studied by MTT colorimetry tests At specified
times (including 24, 48 and 72 h), 110 μl MTT solution (5 mg/ml in PBS)
was added to the wells, cells were incubated further for 1 h at 37 ◦C, and
the resulting formazan salts were dissolved in DMSO (500 μl/well) (Rahnama et al., 2021) The optical density (OD) of each well was recorded using a microplate reader (EZ Microplate Reader 2000, Bio-chrom, Cambridge, UK) at a wavelength of 550 nm after shaking for 10 min Cells not exposed to HGG samples were used as a blank control group, and three independent samples were included for each time in-terval and experimental group
BT474 and HS5 cells were also seeded in 8-well glass-bottom slides (12,000 cells/ml), grown for 24 h and exposed or not to HGG samples
(23.1 % [v/v], also sterilized by UV radiation) for a further 24 h Then,
15 min before imaging the cells by confocal laser scanning microscopy (CLSM), calcein AM (1 μg/ml) and PI (5 μg/ml) were used to stain alive (green) and dead (red) cells, respectively (Huan et al., 2022) Samples (two independent ones for each experimental group) were washed with PBS solution before CLSM imaging (TCS SPS, Leica Microsystems, Wetzlar, Germany)
2.12 HGG loading with PTX:βCD complexes
To improve PTX aqueous solubility, PTX:βCD inclusion complexes were obtained following the freeze-drying method described by Alcaro
et al., 2002 Briefly, PTX (1 mg) was dissolved in absolute EtOH (1.2 ml), and βCDs (1.2 mg) were dissolved in H2O(d) (1.4 ml) Next, the βCD solution was added to the PTX solution, and the resulting hydroalcoholic solution was kept under agitation (100 rpm) for 5 h at room temperature and in the dark Later, it was frozen at − 80 ◦C and freeze-dried (Nieto
et al., 2019) The white powder obtained was dissolved in H2O(d), achieving a 0.185 mM PTX working concentration
Subsequently, to load HGG patches with the PTX:βCDs prepared, hydrogel synthesis was performed as described above PTX:βCD solu-tions were added while the gelation process was taking place, once the L- Cys solutions (3 mg/ml) were incorporated (Ning et al., 2020) HGGs loaded with the chemotherapeutic (HGGs@PTX) were allowed to cool in dishes or multi-well plates for their complete gelation
2.13 PTX-release from HGGs in vitro
Once obtained, crosslinked HGGs@PTX were allowed to gel for 90 min and washed with PBS to remove the unloaded taxane before per-forming drug release experiments in duplicate Next, hydrogel patches (35 mm diameter, 8 mm height) were soaked in crystallizing dishes containing slightly acidic PBS (60 ml, pH 6.8) and incubated at 37 ◦C at
40 rpm for 72 h To mimic the intracellular redox potential of tumor cells, GSH was added in high concentrations (10 mM) to the release medium of some HGG samples (P´erez et al., 2014; Robby et al., 2021) At pre-determined times, 0.5 ml aliquots were taken out, and equal vol-umes of acidic PBS (containing or not GSH [10 mM]) were added to maintain a constant volume in the crystallizing dishes The amount of PTX released was calculated by comparing the absorbance of the ali-quots at 230 nm (UV-1800 spectrophotometer, Shimadzu Corporation, Kioto, Japan) with a previously measured calibration curve obtained from a PTX dilution series Aliquots of the release media of non PTX- loaded HGGs were used as a blank Cumulative PTX release (%) from the different HGGs samples was determined according to Eq (9) and plotted against time (Fang et al., 2021; Rezk et al., 2019; Vu et al.,
2022)
PTX released (%) = Total PTX released
Moreover, PTX release kinetics were studied through four different
mathematical models, i.e., zero-order, first-order, Korsmeyer-Peppas
and Higuchi models A description of the method is reported in the Supplementary Material
Trang 52.14 Antitumor activity of HGGs@PTX in vitro
HGG@PTX antitumor activity was analyzed in vitro on two different
human HER2-overexpressing breast carcinoma cell lines: BT474 and
SKBR3 (Nieto et al., 2019)
Cells were cultured as previously indicated, and MTT assays and
live/death staining were conducted following the same protocols as
before to doubly assess crosslinked HGG@PTX cytotoxicity
Neverthe-less, this time, BT474 and SKBR3 cells were exposed to HGGs (23.1 %
[v/v]), HGGs@PTX (23.1 % [v/v]) and PTX:βCDs (in an equivalent
concentration to that loaded to the HGGs (30.8 μM)) Besides, live/death
staining was performed 48 and 72 h after cell exposure to the different
treatment conditions Again, cells not exposed to HGG samples served as
a blank control group in both assays
In addition, lactate dehydrogenase (LDH) leakage assays were
car-ried out according to LDH activity detection kit manufacturer's
in-structions to analyze BT474 and SKBR3 membrane damage after
treatment with the HGGs[3LCys]@PTX for 48 h Group distributions
and PTX:βCDs and HGG[3LCys]@PTX concentrations similar to those in
the MTT assays were employed The absorbance of the LDH expression
was assessed at 450 nm using a microplate reader
2.15 Statistical analysis
All data were reported as mean ± standard deviation (SD) Specific
comparison between groups was carried out with unpaired Student's t-
tests, while one-way ANOVA was used for multiple-group comparison
p-values <0.05 were considered to be statistically significant When
statistically significant differences were found when performing one-
way ANOVA, Tukey test was carried out as post-hoc analysis
3 Results and discussion
3.1 Preparation of HGGs with different degrees of chemical crosslinking
One of the characteristics of GG that has led to its increased use for biomedical purposes is its ionotropic sensitivity (Das & Giri, 2020;
Palumbo et al., 2020) In this way, obtaining HGGs is possible because, when mono- or divalent cations are present in a solution, GG can un-dergo thermally reversible gelation after transition from a coiled form at
high temperature (>80 ◦C) to a double-helix structure when cooled (Bacelar et al., 2016; Prajapati et al., 2013) Thus, HGG consistency can
be modified, apart from altering the concentration of the gum, by adding different ions to GG solutions (Das & Giri, 2020; Palumbo et al., 2020)
Fig 1 Frequency sweeps of the different synthesized HGGsAB and HGGsPBS performed at 25 ◦C (A–C) and 37 ◦C (B–D) Filled symbols represent G′values, while empty symbols are G′′values The concentration of L-Cys used to crosslink the different hydrogels is indicated between brackets (in mg/ml)
Trang 6For this reason, as indicated in Scheme 1, two buffers of different ionic
composition and pH (AB vs PBS) were used in this work to synthesize
HGGs with the aim of analyzing how they conditioned the
physico-chemical properties of the hydrogels obtained (HGGsAB vs HGGsPBS
respectively) (Matricardi et al., 2009; Oliveira et al., 2016) In addition,
to enhance their stability and make them redox-responsive, HGGs were
chemically crosslinked with L-Cys (Du et al., 2012), which was employed
in three different concentrations (1.5, 3, and 4.5 mg/ml) to later choose
the most suitable hydrogels to act as PTX delivery systems EDC
chem-istry was used to carry out the crosslinking because, unlike other
com-pounds frequently used to prepare chemical hydrogels, EDC and NHS
are not cytotoxic in concentrations below 0.5 M (Hua et al., 2016;
Panczyszyn et al., 2021) In addition, these compounds have already
been used in the literature to crosslink hydrogels made up of other
polymers (Goodarzi et al., 2019; Pacelli et al., 2018; Výborný et al.,
2019), and the N-hydroxysuccinimidyl ester coupling chemistry is one
of the few conjugation strategies utilized in the development of FDA-
approved protein conjugates (Kang et al., 2021; Pelegri-O'Day et al.,
2014)
3.2 Rheological properties of the different HGGs
Once obtained, the viscoelastic properties of the six different
syn-thesized types of HGG were determined employing dynamic oscillatory
frequency sweep assays and compared Mechanical spectra recorded
both at 25 ◦C and 37 ◦C can be found in Fig 1
As can be observed in Fig 1, the frequency sweeps obtained
indi-cated that all samples had characteristic gel behavior, since the storage
modulus (G′) was at least 10 times higher than the loss modulus (G′′) in
all cases Moreover, both G′ and G′′ were almost independent of the
frequency, which is a distinctive fact of entangled gels (Matricardi et al.,
2009; Richa & Choudhury, 2019) However, when comparing the
spectra of the different HGGsAB (Fig 1[A–B]) with those of the HGGsPBS
(Fig 1[C–D]), it was observed that G′ values were greater when
hydrogels were prepared in PBS than in AB In this way, HGGsPBS gelled faster and were more viscous than HGGsAB This result was logical considering that PBS contains K+cations and higher concentrations of
Na+cations (>10 times greater) than AB (Table S1) and, therefore, that
it could contribute to achieving greater degree of GG crosslinking
As expected, when the L-Cys content of both HGGsAB and HGGsPBS
was higher, G′values increased due to the existence of more chemical crosslinkings and the consequent formation of stronger 3D networks This trend could be also seen when increasing the measurement tem-perature from 25 ◦C to 37 ◦C, although this increase in temperature resulted in diminished G′values, which were 40–60 % lower than those recorded at 25 ◦C (Matricardi et al., 2009) Hence, this reduction in the elastic modulus suggested that HGG equilibrium constants were thermal sensitive, and that this sensitivity could be related to the initial degree of crosslinking of the HGGs, since G′reduction was less noticeable when hydrogels were disulfide-crosslinked with higher concentrations of L-Cys and when they were synthesized in PBS instead of in AB (Roberts et al.,
2007)
3.3 Swelling behavior of the different HGGs as a function of the medium
pH and ionic strength
Since the rate and degree of swelling of hydrogels are the most important parameters when controlling the release of the drugs with which they may be loaded (Ganji et al., 2010), the swelling kinetics of all HGGs prepared were analyzed as a function of the medium pH and ionic strength (μ) For this purpose, HGG samples were soaked in H2O(d) and
H2O(c) to determine whether their different ionic composition condi-tioned hydrogel swelling capacity Likewise, HGGs were soaked in NaCl solutions, PBS and supplemented DMEM because these media with different ionic strength mimic physiological fluids Moreover, tris buffer, citrate buffer and AB were also employed to perform swelling assays to try to determine how the medium acidity or basicity could condition HGG absorption capacity The properties of all these media can be found
Fig 2 Swelling kinetics of the different HGGsAB (A–C) and HGGsPBS (D–F) as a function of the swelling time when soaked in solutions with different pH and ionic strength at 25 ◦C
Trang 7in Table S2
The swelling kinetics obtained for the HGGsAB crosslinked with
different concentrations of L-Cys are shown in Fig 2(A–C), while those of
the three different HGGsPBS can be seen in Fig 2(D–F)
As shown in Fig 2, most HGG samples reached equilibrium after 240
min Thereby, after soaking HGGs in the different media for about 4 h,
there was a balance between the osmotic forces caused by the solutions
when entering the hydrogel macromolecular networks and the cohesive-
elastic forces exerted by the GG chains, which opposed the expansion
For this reason, the experimental data obtained up to 240 min were
adjusted to a pseudo-second-order kinetic model to determine Q∞ and
K∞ values for all HGGsAB and HGGsPBS in the different media (Panpinit
et al., 2020; Schott, 1992) The values obtained for these parameters,
which refer to the theoretical equilibrium swelling capacity and the
swelling rate constant of the HGGs, respectively, are indicated in
Table S3 and S4
When comparing the parameters of the swelling kinetics of both
types of HGGs as a function of their crosslinking degree, it was noticed
that, in general, the greater crosslinking, the lower the HGG swelling
capacity This fact was in line with what was expected since by
increasing L-Cys concentration during the synthesis process, it was likely
that HGG pore size would be reduced, and that hydrogels would take up
less volume when soaked in the different media (Coutinho et al., 2010)
In the same way, as the degree of crosslinking of the HGGsAB was
lower than that for the HGGsPBS, they showed greater swelling capacity
and, therefore, higher Q∞ and K∞ values, especially in the most alkaline
media: H2O(d), H2O(c), tris buffer and DMEM Possibly, as described in
the literature, H+cations could interact with GG negative charges after
penetrating the hydrogel structure, causing greater aggregation of GG
chains at low pH values By contrast, in basic media, OH− anions may
accelerate the electrostatic repulsion of GG chains, causing hydrogels to
experience a hydrolysis-induced swelling behavior and to have higher
swelling rates than in acidic solutions (Cassanelli et al., 2018; De Souza
et al., 2016; Moritaka et al., 1995; Zhou & Jin, 2020) In fact, when
HGGs were soaked in H2O(d) and, especially, in tris buffer, they started
to break after 30 min, possibly because the electrostatic repulsion be-tween the COO− anions was too strong and hydrogels lost their network structure In addition, as shown in Fig 2, the less crosslinked HGGsAB experienced over-swelling when soaked in tris buffer, followed by a deswelling process that took place until they reached equilibrium Probably, since these HGGs could oppose less resistance to the entry of tris buffer in their structure, this phenomenon could take place because
of the difference in osmotic pressure that occurred at the initial stage of the swelling process (Li et al., 2021)
Finally, regarding the effect of the ionic strength of the media on HGG swelling behavior, another phenomenon already described in the literature could be observed: in those media with greater ionic strength (DMEM, PBS, citrate buffer, AB and NaCl solution), HGG swelling occurred in a lesser extent than in media with less ions (H2O[d] and H2O [c]) due to GG ionotropic sensitivity Thus, like H+, cations existing in the solutions in which hydrogels were soaked could interact with GG chains, promoting their aggregation and, therefore, lowering HGG me-dium uptake capacity (Coutinho et al., 2010; Moritaka et al., 1995)
3.4 Crosslinking density of the different HGGs
Besides, since crosslinking density (dx) and average molecular weight between crosslinks (Mc) determine hydrogel swelling capacity and, therefore, hydrogel drug release patterns, dx and Mc of the different HGGs were also determined based on the data obtained in the swelling tests once HGGs reach equilibrium in H2O(d) The values calculated for these parameters, as well as for the different polymer volume fractions (Vr), are reported in Table S5
As can be seen in the Supplementary Material, when greater con-centrations of L-Cys were employed for HGG preparation, the average polymer volume fraction and molecular weight between crosslinkings diminished By contrast and as expected, HGG crosslinking density increased In this way, when greater amounts of crosslinker were
Fig 3 Morphological analysis under SEM of (A) HGGAB[1.5LCys], (B) HGGAB[3LCys], (C) HGGAB[4.5LCys], (D) HGGPBS[1.5LCys], (E) HGGPBS[3LCys] and (F) HGGPBS[4.5LCys] samples
Trang 8incorporated, the space for solvent accommodation between GG chains
could be reduced, being this fact in agreement with the results
previ-ously obtained in the swelling tests
3.5 Porosity of the different freeze-dried HGGs
Once the crosslinking degree of the different HGGs was analyzed,
their apparent porosity was calculated and their morphology was
studied by SEM Fig 3 shows the images obtained from all samples once freeze-dried and cross-sectioned, while Table 1 shows HGG mean apparent porosity and the average diameter of the hydrogel macro- and
micropores, determined via image analysis
As can be noticed in both, Fig 3 and Table 1, the macro- and mi-cropores of the HGGAB samples were bigger than those of the HGGPBS
samples, which were less porous In addition, as can be observed in the images, HGGs prepared in PBS had more micropores than those syn-thesized in AB, which again revealed their greater degree of crosslinking
Likewise, regarding the diameter of the macro- and micropores of the HGGs prepared in AB with different concentrations of L-Cys, it should be noted that differences were not statistically significant in the case of macropores, but they were in the case of micropores, since those of the HGGsAB[1.5LCys] were smaller than the micropores of the other
hydrogels according to the post hoc analysis (Tukey test) that was later performed (p < 0.05) On the contrary, the differences in the size of the
macropores of the HGGsPBS were more remarkable than those of the micropores In this manner, the diameter of the micropores of all HGGsPBS was very similar, although as the concentration of L-Cys used in
Table 1
HGG apparent porosity (%) and mean diameter (μm) ± SD of the macro- and
micropores of the different hydrogel samples, once freeze-dried, determined via
SEM image analysis
Sample Porosity (%) Mean macropore size Mean micropore size
HGG AB [1.5LCys] 97.93 ± 1.4 553.1 ± 186.6 μm 325.9 ± 95.5 μm
HGG AB [3LCys] 96.53 ± 2.1 550.3 ± 155.5 μm 215.7 ± 69.0 μm
HGG AB [4.5LCys] 95.88 ± 1.7 561.0 ± 193.7 μm 225.6 ± 32.8 μm
HGG PBS [1.5LCys] 93.01 ± 0.9 442.3 ± 95.9 μm 123.2 ± 48.4 μm
HGG PBS [3LCys] 91.80 ± 1.6 354.3 ± 161.0 μm 123.0 ± 74.7 μm
HGG PBS [4.5LCys] 90.50 ± 1.8 390.0 ± 91.9 μm 127.2 ± 48.0 μm
Fig 4 (A) IR spectra of GG and the different HGGs in the 900–1800 cm− 1 (left) and 1800–4000 cm− 1 (right) ranges; (B) TG curves obtained for GG and the
different HGGs
Trang 9hydrogel synthesis increased, they had greater number of micropores
3.6 Chemical structure of the different HGGs
As can be seen in Fig 4(A), all GG characteristics bands within the
900–4000 cm− 1 range could be distinguished in the spectra of the
different HGGs In this manner, GG-specific peaks were observed at
1032 cm− 1 (–C–O–C– stretching), 1600 cm− 1 (C––O stretching
vi-brations), 2920 cm− 1 (–CH stretching) and 3400 cm− 1 (–OH
stretch-ing) in all samples (Lee et al., 2020) There were no significant
differences between the spectra of the HGGsAB and those of the HGGsPBS
Nevertheless, when comparing GG spectrum to the spectra of the
hydrogels, some alterations (marked in red in Fig 4[A]) could be
appreciated, possibly indicative of HGG successfully crosslinking with L-
Cys via EDC/NHS reaction Herein, HGGs had a peak at 1560–1562 cm− 1
that may correspond to the –CONH– amide bond formation between
GG –COOH and L-Cys –NH groups, and which was not present in GG
spectrum (Panczyszyn et al., 2021) The band at 1375 cm− 1, which
could correspond to the C–H bending and which was marked in the GG
spectrum (Criado et al., 2016), disappeared in the spectra of all HGGs
Finally, the characteristic peak of the -SH group was detected at 2530
cm− 1, and the peaks related to -CH2 vibrations at 2920–2929 cm− 1 were
more pronounced for the HGGs in comparison with GG, which may
confirm the thiolation of the hydrogels after L-Cys crosslinking (George
et al., 2020; Xu et al., 2021)
3.7 Thermal stability of the different HGGs
A TGA of the six different types of HGGs prepared was performed to
evaluate their thermal stability and mass loss and, thus, further
corroborate their crosslinking degree, since differences in degradation
temperatures can give some provision about polymer crosslinking TG
curves obtained for them can be seen in Fig 4(B), along with the GG
curve
As can be noticed in Fig 4(B), both GG and all HGGs showed a two- step thermogram, where the first stage of minor weight loss occurred in the 50–100 ◦C range This weight loss was likely caused by the evapo-ration of the adsorbed buffer/H2O in the samples Thus, it may be directly related to HGG swelling capacity (Ding et al., 2021; Karthika & Vishalakshi, 2015) and, for this reason, it was greater for the HGGAB
samples (11.1–14–4 %) than for the HGGPBS samples (8.5–11.0 %) and
GG (8.6 %) Likewise, HGGs crosslinked with lower L-Cys concentrations lost greater weight than those prepared with higher concentrations of the crosslinker, fact that showed again that L-Cys concentration in samples had an inverse relationship with the swelling capacity of the hydrogels and, consequently, with their porosity
On the other hand, the second stage of weight loss, which occurred in the 250–300 ◦C range, could account for GG degradation and the sub-sequent destruction of the whole hydrogel network structure (Ding
et al., 2021; Karthika & Vishalakshi, 2015) At this stage, HGGAB [1.5L-Cys], HGGAB[3LCys] and HGGAB[4.5LCys] samples lost about 50.6 %,
53 % and 53.7 % of weight, while HGGPBS[1.5LCys], HGGPBS[3LCys] and HGGPBS[4.5LCys] samples lost about 30.8 %, 32.4 % and 33.3 % of weight, respectively Thereby, the overall trend showed that the greater the degree of HGG crosslinking, the smaller their rate of weight loss and the better their thermal stability
3.8 Compression modulus of HGGs[3LCys]
The swelling and deswelling capacity of the hydrogels, which is determined by their crosslinking degree, governs drug release In this way, greater crosslinking degrees reduce hydrogel pore size and desw-elling capacity and decrease the overall diffusion of the drugs through the polymer networks (Khan & Ranjha, 2014; Sivakumaran et al., 2013) Therefore, based on the results obtained up to this point, it was considered that using HGGs[1.5LCys] could lead to a quick burst release
Fig 5 Degradation rate of HGGAB[3LCys] and HGGPBS[3LCys] samples after incubation with lysozyme solutions (1 mg/ml) at 37 ◦C for 9 days
Trang 10of PTX due to their larger pore size (Sivakumaran et al., 2013), while
PTX release from HGGs[4.5LCys] may be too slow because of their
elevated number of micropores Herein, those HGGs crosslinked with 3
mg/ml L-Cys were regarded to be the most suitable hydrogels to achieve
proper, local PTX release, and they were chosen to perform subsequent
assays
Therein, mechanical properties of the HGGs[3LCys] were analyzed
using static compression measurements The average Young's modulus
of both HGGsAB[3LCys] and HGGsPBS[3LCys] was found to be 86.5 ±
12.9 KPa and 95.9 ± 7.8 KPa, respectively Despite being close values (p
> 0.05), slightly increased mechanical strength in HGGsPBS was
ex-pected because of their higher degree of crosslinking In any case, the
compression elastic moduli of both hydrogels were in the range of the
modulus compression elasticity of most biological tissues that are soft
viscoelastic materials (0.1–100 KPa) (Shpaisman et al., 2012), so they
could meet the requirements to potentially be applied in vivo in the
future
3.9 Enzymatic degradation rate of HGGs[3LCys]
Before proceeding to load HGGs[3LCys] with the PTX:βCD com-plexes, their biosuitability was first analyzed using enzymatic degrada-tion assays The results obtained when investigating the degradadegrada-tion behavior of the HGGsAB[3LCys] and the HGGsPBS[3LCys] after incuba-tion with lysozyme soluincuba-tions can be seen in Fig 5
As can be observed in Fig 5, the weight of both hydrogel types decreased gradually with incubation time increasing, which proved their biodegradability Nonetheless, compared to HGGsPBS[3LCys],
Fig 6 (A) Results of the MTT assays performed with HS5 and BT474 cells to assess HGG biocompatibility Cells were exposed to both HGGsAB[3LCys] and HGGsPBS[3LCys] (23.1 % [v/v]), and their relative viability was compared with that of an untreated control The results shown are the average viability values ± SD
of three independent samples; (B) CLSM images of HS5 and BT474 cells 24 h after exposure to HGGs[3LCys] (23.1 % [v/v]) Cell survival and death were assessed by
using calcein AM (green) and propidium iodide (red)